Elsevier

Atherosclerosis

Volume 138, Issue 1, May 1998, Pages 79-89
Atherosclerosis

The differential distribution of hyaluronic acid in the layers of human atheromatic aortas is associated with vascular smooth muscle cell proliferation and migration

https://doi.org/10.1016/S0021-9150(98)00006-9Get rights and content

Abstract

Vascular smooth muscle cells (VSMC), under conditions of induced proliferation, similar to those involved in atherosclerosis, secrete an acidic glycan, 82% of which exhibits structural homology with hyaluronic acid (HA), has a molecular mass of 340 kDa (HA-340) and inhibits VSMC proliferation in vitro. In this study, the expression of glycans was investigated in human atheromatic aortas and evidence is presented that a HA molecule, similar to HA-340, is distinctly expressed in all aortic layers. The isolation of the glycans from human aortas was performed after homogenization of the individual aortic layers (atheromatic plaque, tunica intima, tunica media and tunica adventitia), by lipid extraction and extensive digestion with pronase and DNase. The total glycans were purified from the digestion products by gel filtration on Sephadex G-25 and fractionated on a Superose 6 column. Enzymatic treatment of the ensuing glycan fractions with all known glycosaminoglycan-degrading enzymes, followed by electrophoresis on polyacrylamide gradient gels and cellulose acetate membranes, revealed that, in addition to HA, the tunica intima and the atheromatic plaque also contained dermatan sulfate, while the tunica media and the tunica adventitia also contained chondroitin sulfates and heparan sulfate. The highest concentration of the human aorta HA was found in the tunica media, exhibiting a negative concentration gradient from the tunica media to the atheromatic plaque. Investigation of the biological function of the human aorta HA revealed that this molecule acts as a negative regulator on the PDGF-induced VSMC proliferation and as a positive regulator on the PDGF-induced VSMC migration. The differential expression of HA within the aortic layers correlates with the biological function attributed to this acidic glycan and associates it with key events in the progression of atherogenesis.

Introduction

Atherogenesis, the complex cascade of events leading to the formation of atheromatic plaques, depends on the interaction between several cell types, growth factors, cytokines and molecules of the extracellular matrix [1]. Among different cell types, vascular smooth muscle cells (VSMC) play a major role in the formation of atherosclerotic lesions 2, 3. In the normal human arteries, susceptible to atherosclerosis, VSMC reside mainly in the tunica media in a quiescent state and express a variety of differentiation-specific genes important to maintain the physiological regulation of vessel tone and blood pressure [4]. During the inflammatory injury seen in the early stages of atherosclerosis, VSMC migrate from the tunica media to the tunica intima. These migrating VSMC exhibit a synthetic phenotype with subcellular organization designed to support rapid cell growth and proliferation. Among several growth factors and cytokines platelet-derived growth factor (PDGF) has been shown to be essentially involved in the progression of atherosclerosis [1]. Extensive in vitro studies revealed that PDGF-BB stimulates the proliferation of VSMC, endothelial cells and fibroblasts 1, 5, 6. However, recent data suggest that this growth factor is only a weak mitogen for VSMC in vivo 7, 8, 9but it induces their migration from the tunica media into the tunica intima [1].

Among different molecules involved in atherogenesis the glycosaminoglycans (GAGs) have been reported to contribute in some key events leading to the formation of atherosclerotic lesions [10]. Extracellular matrix GAGs provide structural links between fibrous and cellular elements, contribute to viscoelastic properties, regulate permeability and retention of plasma components within the matrix 10, 11, inhibit vascular cell growth [12], affect hemostasis and platelet aggregation [13]and interact with lipoproteins [14]. Specific GAGs such as dermatan sulfate and chondroitin sulfates A and C have been shown to increase in relation to early fibrous plaque formation in the tunica media [15], the coronary arteries of humans [16], or in the coronary arteries of rhesus monkeys [17]. In the tunica media of atherosclerotic human aortas, an increase in dermatan sulphate and chondroitin sulphate has been reported [18]but others did not observe any changes [19].

Hyaluronic acid (HA), a mainlike GAG, appears to be of particular interest, since tissues enriched in HA may undergo expansion due to the ability of the molecule to bind large amounts of water, thus creating a loose, hydrated micro environment that facilitates cell migration and proliferation, two critical events in atherogenesis [1]and arterial development [20]. In this context, it has been reported that HA is secreted by arterial smooth muscle cells [21], HA-synthesizing enzymes increase during arterial smooth muscle cell proliferation [22]and HA decreases with atherosclerotic involvement of the tunica media 15, 16, 17, 23.

It was recently shown that PDGF-BB specifically stimulates VSMC, in culture, to secrete a 340-kDa acidic glycan molecule (HA-340), 82% of which exhibits structural homology with HA and inhibits VSMC proliferation, in vitro [24]. Here, evidence is presented that the HA which is expressed in all layers of the human aorta and the atherosclerotic plaque has the same molecular mass as that identified in VSMC, in culture. Furthermore, we show that the human aorta HA affects the PDGF-induced VSMC proliferation and migration through an artificial basement membrane (BM), in vitro. The abundance of this molecule in the tunica media as compared with the low levels in the tunica intima and atherosclerotic plaque correlates with its function as a negative control element of VSMC proliferation and as a positive control element of VSMC migration during the development of atherosclerotic lesions.

Section snippets

Isolation and purification of glycans

Biopsies of human aortas (upper thoracic level and its adjacent part of the aortic arch), from male adults (n=4, age 30–35 years) were obtained at autopsy within 6 h of death by accident and were kindly provided by the Department of Forensic Medicine, School of Medicine, Aristotle University of Thessaloniki, Greece. The medical history of the individuals was free from diseases, such as hypertension and diabetes, but autopsy revealed that there was significant plaque development in their aorta.

Isolation and purification of the total glycans

Human atheromatic aortas, obtained at autopsy, were dissected in the atheromatic plaque, the tunica intima, the tunica media and the tunica adventitia. The atherosclerotic lesions were identified as fibrofatty. Total glycans were isolated after homogenization of tissue samples, delipidation and sequential treatment with pronase, DNase and alkali borohydride. Purification of the glycans from the digestion products was achieved on a Sephadex G-25 column as a single peak, with 90% recovery (data

Discussion

GAGs are a group of complex macromolecules that are expressed in a wide range of tissues and compose an essential part of the extracellular matrix. They also play a pivotal role in sequestering and presenting a wide range of growth factors and cytokines to a diverse range of responding cell types [36]. We have recently reported that PDGF specifically stimulates proliferating VSMC to secrete HA-340, a 340-kDa HA-like molecule [24]. This increased production of HA-340 was restricted to

Acknowledgements

E. Papakonstantinou was supported by a fellowship from the European Commission (ERBFMBI-CT-96-0871).

References (48)

  • G.N. Misevic

    Immunoblotting and immunobinding of acidic polysaccharides separated by gel electrophoresis

    Methods Enzymol

    (1989)
  • R. Hata et al.

    A rapid and micro method for the separation of acidic glycosaminoglycans by two-dimensional electrophoresis

    Anal Biochem

    (1972)
  • B.A. Mast et al.

    Hyaluronic acid modulates proliferation, collagen and protein synthesis of cultured fetal fibroblasts

    Matrix

    (1993)
  • G.A.A. Ferns et al.

    Hyaluronan (Hyal-BV 5200) inhibits neo-intimal macrophage influx after balloon-catheter induced injury in the cholesterol-fed rabbit

    Atherosclerosis

    (1995)
  • R. Ross

    The pathogenesis of atherosclerosis: a perspective for the 1990s

    Nature

    (1993)
  • R. Ross et al.

    Cellular interactions, growth factors and smooth muscle proliferation in atherogenesis

    Ann NY Acad Sci

    (1990)
  • H.C. Stary

    Composition and classification of human atherosclerotic lesions

    Virchows Arch A Pathol Anat Histopathol

    (1992)
  • M.W. Majeski et al.

    Platelet-derived growth factor (PDGF) ligand and receptor gene expression during repair of arterial injury

    J Cell Biol

    (1990)
  • H. Okazaki et al.

    Regulation of platelet-derived growth factor ligand and receptor gene expression by a-thrombin in vascular smooth muscle cells

    Circ Res

    (1992)
  • A. Jawien et al.

    Platelet-derived growth factor promotes smooth muscle migration and intimal thickening in a rat model of balloon angioplasty

    J Clin Invest

    (1992)
  • G.A.A. Ferns et al.

    Inhibition of neointimal smooth muscle accumulation after angioplasty by an antibody to PDGF

    Science

    (1991)
  • C.L. Jackson et al.

    Role of endogenous platelet-derived growth factor in arterial smooth muscle cell migration after balloon catheter injury

    Arterioscler Thromb

    (1993)
  • T.N. Wight

    Cell biology of arterial proteoglycans

    Atherosclerosis

    (1989)
  • T.N. Wight

    Proteoglycans in pathological conditions atherosclerosis

    Fed Proc

    (1985)
  • Cited by (0)

    View full text